This invention is related to a method for measuring the concentration of protease inhibitors, a kit for use in such a method and a method for dissolving a substrate.
Trypsin inhibitors in urine such as urinary trypsin inhibitor (UTI) have lately attracted considerable attention as an indicator for the condition of an organism, and research has been carried out in the field of clinical medicine. For example, it is known that UTI is found in urine in an organism when it is under internal or external stresses caused by inflammation, surgical operation, or the like ("The Clinical Importance of Urinary Trypsin Inhibitor and its clinical usefulness for diagnosis of acute phase reactant and renal disease" Shiro Kuwajima et al., JAPANESE JOURNAL OF INFLAMMATION REVIEW ARTICLE, VOL. 9, NO. 3, MAY 1989).
Because the inhibitory activity of the urinary trypsin inhibitor depends on the amount present, its concentration is evaluated by measuring the inhibited activity of trypsin. The measurement is carried out, for example, by mixing a urine sample, an enzyme (i.e. protease) solution containing trypsin, and a buffer solution, and then adding a substrate solution to the mixture and measuring the enzyme reaction.
In this measurement, benzoyl-arginine-p-nitroanilide (BAPNA) can be used as a substrate. However, because BAPNA is only slightly soluble in water, this substrate solution is prepared by dissolving BAPNA in dimethyl sulfoxide (DMSO) and then diluting the mixture about two-fold with water. Furthermore, calcium is normally used as a trypsin activation agent in this measurement, and it is usually mixed in the buffer solution.
However, there are problems, described below, in such a conventional method for measurement.
First, when the concentration of the calcium mixed in the buffer solution or the like is low, trypsin may be activated by the influence of calcium present in the urine sample, so that the observed trypsin activity measurement would indicate a lower value for the urinary trypsin inhibitor concentration than the real value. Furthermore, if an excess amount of calcium is added, it reacts with carbonate ions, phosphate ions and the like present in the urine to produce precipitates, which affect the measurement. Although pretreatment such as centrifugation or the like may be conducted to remove them, this may complicate the measurement.
Furthermore, an organic solvent such as DMSO may damage a plastic cell generally used in an automatic analytical apparatus, so that the amount of the organic solvent which can be used is limited. Accordingly, the amount of the substrate which can be dissolved in the organic solvent is also limited. As a result, the sensitivity of the measurement is difficult to improve, and the reproducibility is limited. Moreover, there is a possibility of trypsin activity being inhibited by using an organic solvent. In addition, the rather insoluble BAPNA can be dissolved by using an organic solvent, but if the amount used is not sufficient, there is a possibility of BAPNA crystallizing out of solution when the substrate solution is kept in long-term storage or in refrigeration. Therefore, in a conventional measuring method, when a slightly soluble substrate such as BAPNA is used with an organic solvent, it has been necessary to prepare a substrate solution at each measurement, and immediately thereafter provide it for the measurement.